Microfluidic devices for the controlled manipulation of small volumes

ABSTRACT

A method for conducting a broad range of biochemical analyses or manipulations on a series of nano- to subnanoliter reaction volumes and an apparatus for carrying out the same are disclosed. The invention is implemented on a fluidic microchip to provide high serial throughput. In particular, the disclosed device is a microfabricated channel device that can manipulate nanoliter or subnanoliter reaction volumes in a controlled manner to produce results at rates of 1 to 10 Hz per channel. The reaction volumes are manipulated in serial fashion analogous to a digital shift register. The invention has application to such problems as screening molecular or cellular targets using single beads from split-synthesis combinatorial libraries, screening single cells for RNA or protein expression, genetic diagnostic screening at the single cell level, or performing single cell signal transduction studies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/373,129,filed Feb. 24, 2003, now U.S. Pat. No. 7,238,268 which is a continuationapplication Ser. No. 09/408,060, filed Sep. 29, 1999, now U.S. Pat. No.6,524,456 which in turn claims the benefit of priority of U.S.Provisional Application 60/148,502, filed on Aug. 12, 1999, . Each ofthe foregoing applications is hereby incorporated herein by reference.

The United States Government has rights in this invention pursuant tocontract no. DE-AC05-00OR22725 between the United States Department ofEnergy and UT-Battelle, LLC.

FIELD OF THE INVENTION

This invention relates to a microfabricated fluidic device, and inparticular, to such a device that is configured for forming andtransporting a series of minute volume segments of a material and forstorage, retrieval, and analysis thereof and to a method for forming,transporting, storing, and retrieving such a series of minute volumesegments.

BACKGROUND OF THE INVENTION

A number of elementary microfabricated fluidic devices have beendemonstrated over the past few years. Although many of these fluidicdevices are quite simple, they are demonstratively powerful inaddressing many realistic applications and may well revolutionize theway that biochemical separations are performed. The majority of thedemonstrations have involved transferring known chemical measurementtechniques, such as electrophoresis or liquid chromatography, ontomicrofabricated platforms. Such demonstrations suggest thatmicrofabricated separation devices will be quite useful for improvingthe time and cost associated with collecting information from suchexperiments. However, the known devices have not exploited the newexperimental approaches that such microfabricated devices potentiallyenable. We believe that through improvements in microfluidic control,new more powerful biochemical experimental paradigms will arise.

The area of microfabricated fluidics that has received the mostattention is electrokinetically driven processes. Electrokinetic fluidmanipulations have been demonstrated for mixing and reacting reagents,injection or dispensing of samples, and chemical separations.Electrically driven separation techniques such as capillaryelectrophoresis (CE), open channel electrochromatography (OCEC) andmicellar electrokinetic capillary chromatography (MEKC) have beendemonstrated by a number of research groups. Both dsDNA fragments andsequencing products have been sized using microchip capillary gelelectrophoresis coupled with laser induced fluorescence detection. Lessconventional electrophoretic separations have been studied in postarrays using DC and pulsed electric fields. In additionfluorescence-based competitive immunoassays have been demonstrated usingmicrochip electrophoretic separation of bound and free labeled antigen.These miniature devices have shown performance either equivalent to orbetter than conventional laboratory devices in all cases investigatedand appear to offer the rare combination of “better-faster-cheaper”simultaneously. Microchip separation devices exhibit speed advantages ofone to a few orders of magnitude over conventional approaches. Theefficiency of electrophoretic separations under diffusion limitedconditions is proportional to the voltage drop experienced by thesample. These diffusion limiting conditions can be achieved for shortseparation distances on microchips due to the narrow axial extent of theinjection plugs that are generated. The time of analysis decreasesquadratically with separation distance at constant applied potential,which gives a fundamental advantage to microchip-based electrophoreticseparations.

Other significant advantages of microchip based chemical separations arethe small volumes that can be analyzed, the ability to monolithicallyintegrate sample processing and analysis, and the low cost ofreplication which makes possible highly parallel analyses. All of thesefactors are consistent with high throughput analysis and reductions incost and time to generate biochemical information. Early effortsdemonstrating integration of sample processing include post-separationand pre-separation derivatization of amino acids coupled toelectrophoretic separations. On-chip DNA restriction digestions and PCRamplifications have been coupled with electrophoretic fragment sizing onintegrated monolithic microchips. Cell lysis, multiplex PCR, and CEanalysis were performed on plasmic-containing E. coli cells in a singledevice. Parallel PCR/CE assays of multiple samples in chips containingmultiple reaction wells have also been demonstrated. In addition,competitive immunoassay experiments have been performed on a microchipdevice that included fluidic elements for mixing of sample withreagents, incubation, and electrophoretic separations. Othermicrofabricated fluidic elements that have been coupled to electricallydriven separations include electrospray ionization for analysis by massspectrometry, and sample concentration using porous membrane elementsand solid phase extraction. Devices have also been demonstrated thatemploy electrokinetic transport solely for performing chemical andbiochemical reactions. Examples include devices for enzymatic reactionkinetics, enzyme assays, organic synthesis, and cellular manipulations.All four of these latter applications could eventually be of significantimportance to experimental biology, but have not been sufficientlydeveloped at this time.

A number of microfabricated fluidic devices have also been demonstratedthat use hydraulic forces for fluid transport. While the use ofhydraulic forces can be applied to a broader range of fluidic materialsthan electrokinetic phenomena, it is less convenient to implement ingeneral. External connections to microchips for hydraulically drivenflow are more cumbersome than applying an electric potential. Moreover,electrokinetically driven forces follow the flow of electrical currentand thus, allow greater control over transport within a microchannelmanifold versus application of pressure or vacuum to a terminus of sucha manifold. Electrokinetic forces are also able to generate much highereffective pressures than is practical with hydraulics. The demonstratedcapabilities of hydraulically driven devices appear to be trailing thatof electrokinetically driven devices. Nonetheless, a number of importantcapabilities have been reported.

Microfluidic devices for performing PCR have received considerableinterest. Early devices included only chambers machined in silicon toact as sample reservoirs while later devices utilized the siliconstructure for resistive heating or utilized integrated filters for theisolation of white blood cells. More recently, an interesting device forcontinuous flow PCR was reported that utilized a single microchannelthat meanders through temperature zones to accomplish thermal cycling.Filters for processing cellular materials have been micromachined intosilicon substrates. Flow cytometry devices have also been micromachinedinto silicon and glass substrates and driven hydraulically.

The capability to manipulate reagents and reaction products “on-chip”suggests the eventual ability to perform virtually any type of“wet-chemical” bench procedure on a microfabricated device. The paradigmshift of moving the laboratory to a microchip includes the advantages ofreducing reagent volumes, automation or material manipulation with nomoving parts, reduced capital costs, greater parallel processing, andhigher processing speed. The volume of fluids that are manipulated ordispensed in the microfluidic structures discussed above is on thenanoliter scale or smaller versus tens of microliters at the laboratoryscale, corresponding to a reduction of three orders of magnitude ormore. Flow rates on the devices that have been studied are of the orderof 1 mL/yr of continuous operation. By implementing multiple processesin a single device (vertical integration), these small fluid quantitiescan be manipulated from process to process efficiently and automaticallyunder computer control. An operator would only have to load the sampleto be analyzed. Obviously, this serial integration of multiple analysissteps can be combined with parallel expansion of processing capacity byreplicating microfabricated structures, e.g., parallel separationchannels, on the same device.

Although the so-called “Lab-on-a-Chip” appears to hold many promises,and it is believed that it will fulfill many of them, there are furtherdevelopments necessary to achieve an impact level that parallels thescale of miniaturization realized in the field of microelectronics.There are at least four significant issues that must be addressed tobring “Lab-on-a-Chip” devices to the next level of sophistication, orprocessing power, over the next decade. Those issues are: advancedmicrofluidic control, the “world-to-chip” interface, detection, andviable manufacturing strategies. Presently, electrokinetic manipulationof fluids in microchannel structures represents the state-of-the-art incontrolled small volume handling with high precision. The strategy hasbeen to control the time-dependent electric potential at each of thechannel terminals to move materials along a desired path. While thisstrategy has been very effective for valving and mixing in simpledesigns, it is limited in its applicability and performance as designsbecome more complex. We believe that new strategies that allow controlof electric potentials at multiple points in the microchannel designwill be necessary for these more complex structures. Electrokinetictransport also has limitations in the types of solutions and materialsthat can be manipulated.

The world-to-chip interface is the term we have assigned to the problemof delivering multiple samples or reagents onto microchips to achievehigh throughput analysis. Although a given sample can be analyzed intimes as brief as a millisecond, multiple samples cannot presently bepresented to microchip devices at such a rate. There has been verylittle effort directed toward this problem, but it represents a majorhurdle to achieving ultrahigh throughput experimentation.

SUMMARY OF THE INVENTION

In accordance with a first aspect of the present invention, there isprovided a method of forming and transporting a series of minute volumesegments (nanoliter or subnanoliter) of a material on a fluidicmicrochip, wherein each of the volume segments are separated bysegmenting material. The method includes the steps of providing a firstchannel having an inlet end connected to a source of transport fluid andan outlet end connected to a fluid reservoir and providing a secondchannel having an inlet end connected to a source of segmenting fluidand an outlet end interconnected with said first channel. A minutevolume of the segmenting fluid is drawn into the first channel andtransported in the first channel toward the fluid reservoir. The stepsof drawing and transporting the minute volume of segmenting fluid intothe first channel are repeated to form a series of transport fluidvolumes and/or analysis volumes between the segmenting fluid volumes.Reagents, cells, or reaction beads can be injected or inserted into thetransport fluid volumes to provide a series of assay or analysisvolumes. The assay or analysis volumes are transported in series fashionso as to provide serial registration thereof for storage and retrievalfor later analysis or other manipulation on the microchip.

In accordance with another aspect of this invention, there is providedan apparatus for forming and transporting a series of minute volumesegments of a material. The device is a fluidic microchip having firstand second microchannels formed on a substrate. The first microchannelhas an inlet end connected to a source of transport fluid and an outletend connected to a fluid reservoir. The second microchannel has an inletend connected to a source of segmenting fluid and an outlet endinterconnected with the first microchannel. A plurality of electrodesare disposed in the first microchannel between the outlet ends of thefirst microchannel and the second microchannel. A second plurality ofelectrodes are disposed in said first microchannel between the inlet endof said first microchannel and said second microchannel. The devicefurther includes means for (i) inserting a volume of the segmentingfluid from the second microchannel into the first microchannel, therebydisplacing the transport fluid contained in the first microchannel, (ii)stopping the transport of the segmenting fluid volume in the firstmicrochannel, and then (iii) transporting the interleaved transport andsegmenting volumes in the first microchannel toward the fluid reservoir.

Further embodiments of the device according to this invention includeadditional channels, sources of reagents, reagent diluents, cells,and/or reaction particles, for inserting such materials into transportvolumes formed by sequential pairs of the segmenting fluid volumes.Means for transporting the reagents, cells, and/or reaction beads arealso included in such embodiments. In a preferred arrangement, the firstmicrochannel includes one or more loops to provide serial storage of thereaction volumes for later retrieval and analysis or manipulation.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, and the following detailed description, will bebetter understood when read in conjunction with the attached drawings,in which:

FIGS. 1A, 1B, and 1C are partial schematic diagrams of a microchipshowing the sequence of steps in inserting a segmenting fluid volumeinto a microchannel containing a transport fluid in accordance with oneaspect of this invention;

FIG. 2 is a partial schematic diagram of a microchip showing the loadingof a reagent into transport volumes between alternating pairs ofsegmenting fluid volumes in a microchannel in accordance with anotheraspect of this invention;

FIG. 3 is a partial schematic diagram of a microchip showing the loadingof particles between alternating pairs of segmenting fluid volumes in amicrochannel in accordance with another aspect of this invention;

FIG. 4 is a partial schematic diagram of a microchip showing thesequence of inserting a segmenting fluid, an enzyme, and a substrateinto a microchannel in series fashion in accordance with a furtheraspect of this invention;

FIG. 5 is a partial schematic diagram of a microchip showing thesequence of inserting a segmenting fluid, a reaction particle, and areagent fluid into a microchannel in series fashion in accordance with astill further aspect of this invention;

FIG. 6 is a partial schematic diagram of a microchip showing anarrangement for preparing a series of enzyme-bead assays;

FIG. 7 is a schematic diagram of a microchip showing a arrangement forstoring and retrieving a series of cells, reaction beads, or reagentvolumes; and

FIG. 8 is a schematic of a system for investigating and identifying newdrugs which incorporates a microchip as shown in FIG. 7.

DETAILED DESCRIPTION

Referring now to the drawings, wherein like reference numerals refer tothe same or similar components or features, and in particular to FIGS.1A, 1B, and 1C, there is shown a main microchannel 10. Main microchannel10 is substantially linear and has an inlet 12 that is connected to asource of transport fluid (not shown) and an outlet 14 that is connectedto a waste reservoir (not shown). A branch channel 16 has an inlet 18and an outlet 20 that intersects with the main microchannel 10 forconducting a segmenting or isolating fluid 22 into the main channel 10.Electrodes e1, e2, e3, and e4 are disposed at spaced apart locationsalong main microchannel 10 between outlet 20 and outlet 14. Electrodese5, e6, and e7 are disposed at spaced apart location along the side ofmain microchannel between outlet 20 an inlet 12. All electrodes are incontact with fluids contained within the microchannels. Either thetransport fluid, the segmenting fluid, or both are transportable throughthe microchannels when exposed to an axial electric field. This functionwill be referred to as electrokinetic flow and includes such phenomenaas electrophoresis, electroosmosis, and electrohydrodynamic transport.

With the arrangement shown in FIGS. 1A, 1B, and 1C, the segmenting fluid22 can be inserted into the main microchannel 10 as a series ofdiscrete, minute volumes. The steps for accomplishing the insertion ofthe minute volumes of the segmenting fluid 22 are essentially asfollows. Main microchannel 10 is filled with the transport fluid. Asource of electrical potential 24 a is applied between electrodes e1 ande2 and a second source of electrical potential 24 b is applied betweenelectrodes e6 and e7. The magnitudes and polarities of the electricpotentials are selected to induce electrokinetic flow of the transportfluid in main microchannel 10 in the directions indicated by the arrowsin FIG. 1A. Such flow of the transport fluid causes the segmenting fluid22 to be drawn into main microchannel 10 as shown in FIG. 1B. Assumingthat the segmenting fluid has a lower conductivity than the transportfluid, when the segmenting fluid 22 comes into contact with electrodee7, the current between electrodes e6 and e7 drops, and fluid flow inthat direction ceases. However, the segmenting fluid 22 continues toflow toward the outlet 14 by virtue of the electrical potential acrosselectrodes e1 and e2. When the desired volume of segmenting fluid hasbeen drawn into main microchannel 10, the volume of segmenting fluid isdispensed into main microchannel 10 by applying a source of electricpotential 24 c between electrodes e5 and e6. The magnitude and polarityof the electric potential between electrodes e5 and e6 are selected toinduce electroosmotic flow of the transport fluid in main microchannel10 in the directions indicated by the arrows in FIG. 1C. When the volumeof segmenting fluid comes into contact with electrode e1, the currentbetween electrodes e1 and e2 drops, and fluid flow in that directionceases. An electric potential is then applied between electrodes e2 ande3 to continue transporting of the transport fluid and the segmentingfluid volume. Similarly, when the volume of segmenting fluid comes intocontact with electrode e2, the current between electrodes e2 and e3drops, and fluid flow in that direction ceases. An electric potential isthen applied between electrodes e3 and e4 to further continue thetransporting of the transport fluid, and the segmenting fluid volume,along main microchannel 10. The physical construction and operation of alinear pumping arrangement of the type used in the present invention isdescribed in greater detail in our copending patent application Ser. No.09/244,914 the entire specification of which is incorporated herein byreference.

As an alternative to electrokinetic transport mechanisms, the moving ofthe transport fluid and the injection of the segmenting fluid, and anyother materials used in a device or method according to the presentinvention can be accomplished by application of pressure or vacuum tothe appropriate channel or channels. It is also contemplated thatcombinations of electrokinetic, pressure, and/or vacuum transportmechanisms can be utilized in implementing a given device or method inaccordance with this invention, if desired.

After a segmenting volume has traveled a sufficient distance in mainmicrochannel 10, the process is repeated to insert another segmentingvolume. If the pumping rate of the segmenting fluid into the mainmicrochannel 10 is not sufficiently high, then similar electrodes can beplaced in branch channel 16.

The segmenting fluid is preferably a liquid that is immiscible in thetransport fluid and the reaction fluid(s). Also, the segmenting fluidshould be biocompatible with biological reagents that are used. Thesegmenting fluid is preferably nonconducting for operational control ofthe reaction/transport process. For example, a nonconducting fluidprovides a convenient way to track the location of reaction and pumpingvolumes in the series of minute volumes transported in the microchannel.Further, the segmenting fluid should have a minimal chemicaldistribution coefficient for the various reagents that are used.Paraffin and mineral oils are suitable because they have been used toisolate cells in small volumes of extracellular solutions without anydeleterious effects. Perfluorocarbons may also be suitable because theyare widely used where biocompatibility is required. Silicon oils are yetanother suitable class of materials for the segmenting fluid. Gases suchas propane may be suitable for use as the segmenting fluid, but could bedissolved into the transport or reaction fluid or escape through agas-permeable cover plate, thereby reducing their effectiveness forisolating fluid segments.

Following are descriptions of preferred arrangements for implementingvarious manipulations of minute amounts of materials in serial fashionon a fluidic microchip. Although not shown or described in connectionwith the various embodiments, the various configurations can includearrangements of electrical contacts as described with reference to FIG.1 to provide the electric potentials necessary to effectuate thetransport of the fluidic materials in the channels of variousmicrofabricated devices. Alternatively, as described above, pressure orvacuum means can also be utilized.

A significant feature of the method according to the present invention,is the capability of inserting a series of minute reaction fluid volumesinto the series of minute volume segments in the main microchannel.Referring now to FIG. 2, there is shown an arrangement for thecontrolled loading of reagents into a series of reaction volumes in mainmicrochannel 10. A plurality of segmenting fluid volumes 26 a, 26 b, 26d, and 26 e are inserted into main microchannel 10 at spaced intervalsas described above. A reagent channel 28 has an inlet 30 and an outlet32 that intersects with the main microchannel 10 for conducting achemical or biochemical reagent 34 into the main microchannel 10. Awaste channel 38 interconnects with main microchannel 10 at a locationsubstantially opposite the outlet 32. A diluent channel 40 interconnectswith the reagent channel 28 so that a dilution agent can be mixed intothe reaction fluid 34. The steps for accomplishing the insertion ofminute volumes of the reaction fluid 34 are essentially as follows.

The transport fluid and the segmenting fluid volumes 26 a, 26 b, 26 c,26 d, and 26 e are pumped through main microchannel 10. When asequential pair of segmenting fluid volumes 26 b and 26 c are adjacentthe outlet 32, the pumping is stopped and the reaction fluid 34 ispumped into the volume between segmenting fluid volumes 26 b and 26 c.The transport fluid contained between the segmenting fluid volumes 26 band 26 c is preferably conducted into the waste channel 38.Alternatively, the transport fluid can be added to or replaced. Thereaction fluid volume 36 a is then transported electrokinetically alongmain microchannel 10. Although the process according to this aspect ofthe invention has been described with a static mode of operation, itwill be recognized that dynamic transfer of the reaction fluid into thereaction volumes will provide higher throughput. Such dynamic operationcould be implemented by controlled transport of the transport fluid andthe reaction fluid such that the reaction fluid is injected insynchronism with the arrival of a reaction volume at the outlet 32.

Preferably, the reaction fluid volumes 36 a and 36 b are containedbetween alternate sequential pairs of segmenting fluid volumes. Thus, asshown in FIG. 2, reaction fluid volume 36 a is contained betweensegmenting fluid volumes 26 b and 26 c. Whereas, reaction fluid volume36 b is contained between segmenting fluid volumes 26 d and 26 e. Suchalternate sequencing permits the pumping of the reaction fluid volumesthrough the main microchannel without exposing the reaction fluid to anaxial electric field, or in cases where the reaction fluid does notsupport electrokinetic flow, transport is effected. In other words, theelectric potentials are applied only to the segments containingtransport fluid. The concentration of the reaction fluid is adjusted bymixing a diluent into the reagent before it is injected into the volumebetween the segmenting volumes. In this manner, a series of reagentvolumes each having a different concentration can be generated.

Another significant feature of the method according to the presentinvention is the capability to insert a series of reaction particles,such as beads or cells, into the series of minute volume segments in themain microchannel. Referring now to FIG. 3 there is shown an arrangementfor sorting and loading a plurality of reaction particles 44 into aseries of reaction volumes in main microchannel 10. A plurality ofsegmenting fluid volumes 26 a, 26 b, 26 c, 26 d, and 26 e are drawn intomain microchannel 10 at spaced intervals as described above. A particlereservoir 42 contains a plurality of reaction particles 44 in asuspension fluid. A particle sorting channel 46 is connected to theoutlet of the reservoir 42 for receiving the particles 44. A pair offocusing channels 48 a and 48 b interconnect with the particle sortingchannel 44. The focusing channels 48 a and 48 b provide a focusing fluidto narrow down the flow of particles 44 to a single-particle-widthstream. Electrokinetic focusing of this type is described in ourco-pending patent application Ser. No. 09/098,178, and our issued U.S.Pat. No. 5,858,187, the entire specifications of which are incorporatedherein by reference.

A reaction particle channel 50 intersects the particle sorting channel46 at a location downstream of the focusing channels 48 a and 48 b. Atthe intersection of the reaction particle channel 50 and the particlesorting channel 46, the desired reaction particles 44′ are separatedfrom the non-desired particles 44″. A potential or pressure is appliedto the inlet 50 a of channel 50 to direct particles 44′ into and alongchannel 50 to the main microchannel 10. Reaction particle channel 50 hasan outlet 56 that interconnects with the main microchannel 10 forconducting the reaction particles into the main microchannel 10. Theundesired particles 44″ are conducted away along a particle wastechannel 52 that extends from the particle sorting channel 46.

The steps for accomplishing the insertion of the reaction particles 44′into the transport stream are essentially as follows. The transportfluid and the segmenting fluid volumes 26 a, 26 b, 26 c, 26 d, and 26 eare pumped electrokinetically through main microchannel 10. When asequential pair of segmenting fluid volumes 26 b and 26 c are adjacentthe outlet 56, the particle suspension fluid with a single particle ispumped electrokinetically into the volume between segmenting fluidvolumes 26 b and 26 c, and the transport fluid contained therein isconducted into the waste channel 38. In this arrangement, the wastechannel cross section, or at least its inlet, is sized to prevent theparticle from passing through. The reaction particle and its volume ofsuspension fluid is then transported electrokinetically along mainmicrochannel 10 to a detection/analysis channel 39. Preferably, thereaction particles are contained between alternate sequential pairs ofsegmenting fluid volumes. Thus, as shown in FIG. 3, a first particle iscontained between segmenting fluid volumes 26 b and 26 c. Whereas, asecond particle is contained between segmenting fluid volumes 26 d and26 e. Such alternate sequencing permits the pumping of the reactionfluid volumes through the main microchannel without exposing thereaction particle to an axial electric field. To that end, the electricpotentials are applied only to the segments containing transport fluid.

The ability to precisely manipulate fluid flow and reagent mixing with afluidic microchip lends itself to the study of enzymatic activity andinhibition thereof. An enzyme assay microchip has important implicationsfor drug discovery and medical diagnostics. Referring to FIG. 4, thereis shown an arrangement for providing high throughput enzyme assays. Aplurality of segmenting fluid volumes 26 a, 26 b, 26 c, and 26 d areinserted into main microchannel 10 at spaced intervals as describedpreviously in this specification. An enzyme channel 428 has an inlet 430and an outlet that intersects with the main microchannel 10 forconducting a fluidic enzyme material 434 into the main microchannel 10.A waste channel 38 interconnects with main microchannel 10 at a locationsubstantially opposite the outlet of enzyme channel 428. A diluentchannel 440 interconnects with the enzyme channel 428 so that a dilutionagent can be mixed into the enzyme material 434. A substrate channel 68has an inlet and an outlet that intersects with the main microchannel 10downstream of the enzyme channel outlet for conducting a fluidicsubstrate material 70 into the main microchannel 10. A diluent channel72 interconnects with the substrate channel 68 so that a dilution agentcan be mixed into the substrate material 70.

The steps for accomplishing the insertion of minute volumes of theenzyme material 434 into the main microchannel are essentially the sameas those described for injecting the reagent fluids with reference toFIG. 2. The transport fluid and the segmenting fluid volumes 26 a, 26 b,26 c, 26 d, and 26 e are pumped electrokinetically through mainmicrochannel 10. When an enzyme volume segment 434 a contained between asequential pair of segmenting fluid volumes 26 b and 26 c is adjacentthe outlet of substrate channel 68, the pumping is stopped and thefluidic substrate material 70 is pumped into the enzyme volume segment434 a. The enzyme material and the substrate are mixed in the reactionvolume. The combined fluid volume is then transported along mainmicrochannel 10 to a detection/analysis channel 39. The concentrationsof the enzyme and substrate materials can be varied by varying theamount of diluent mixed into each one, thereby producing a multitude ofdifferent enzyme assays which can be transported along the microchannel10 in serial fashion.

Means for adding an inhibitor to the reaction volume can also beprovided. In one embodiment of such a process, a bead shift register isimplemented for delivery of the inhibitors. In such an arrangement, theenzyme, substrate, and inhibitor will be reacted, and the location ofthe bead leading to positive inhibition is recorded for future analysis.An alternative arrangement is to pool a bead library into a reservoirfrom which the beads are randomly dispensed. Individual beads aretransported to a location where a compound is delivered to a reactionvolume containing an assay target. The bead is indexed in a shiftregister arrangement such as that shown and described with reference toFIG. 3, but only for a time sufficient to give an assay result. If theresult is negative, the bead is transferred into a general storagereservoir, but if inhibition is observed, the bead is stored in theshift register for either immediate or later identification of thecompound, e.g., by electrospray mass spectrometry.

The enzyme activity of the reaction volume is analyzed with appropriateinstrumentation in the analysis channel 39 which is located at anappropriate distance downstream from microchannel 10. The actualdistance depends on the required incubation time of the enzyme,substrate, and inhibitor, and the average linear velocity of the assayreaction volume.

In one example of an enzyme assay using electrokinetic mixing andtransport of reagents on a fluidic microchip in accordance with thisembodiment of the present invention, a fluorogenic substrate(resorufin-β-D-galactopyranoside) is mixed with the enzymeβ-galactosidase. The kinetics of the reaction are obtained by monitoringthe fluorescence of the hydrolysis product, resorufin. Michaelis-Mentenconstants are derived for the hydrolysis reaction in the presence andabsence of inhibitors. A second example of an enzyme assay that can beimplemented in the method according to the present invention, is anassay for determining acetylcholinesterase (AchE) activity. This is atwo stage assay whereby the AchE hydrolyzes acetylthiocholine tothiocholine which in turn reacts with coumarinylphenylmaleimide (CPM) toproduce a highly fluorescent thioether, CPM-thiocholine. Thefluorescence of the latter product is monitored to determine the enzymeactivity. The presence of an inhibitor would reduce the fluorescencesignal relative to the absence of such inhibitor.

Shown in FIG. 5 is an arrangement for high throughput screening forcellular assays including cell viability. A plurality of segmentingfluid volumes 26 a-26 g are inserted into main microchannel 10 at spacedintervals from the branch channel 16 as described above. A cell channel50 intersects the main microchannel 10 for conducting the cells 44′ intothe main microchannel 10 at a location downstream of the branch channel16. The cells are suspended in a biocompatible buffer. A waste channel38 interconnects with main microchannel 10 at a location substantiallyopposite the outlet of cell channel 50. The cross section of wastechannel 38 is dimensioned to be smaller than the minimum majorcross-sectional dimension of the cells 44′. A reagent channel 28intersects the main microchannel 10 at a location downstream of the cellchannel 50. A second waste channel 38′ interconnects with mainmicrochannel 10 at a location substantially opposite the outlet ofreagent channel 28.

The transport fluid and the segmenting fluid volumes 26 a-26 g arepumped through main microchannel 10. When a sequential pair ofsegmenting fluid volumes 26 b and 26 c are adjacent to the outlet ofcell channel 50, the cell suspension fluid is pumped into the reactionvolume between segmenting fluid volumes 26 b and 26 c so that a singlecell is inserted into the reaction volume. Alternatively, an ensemble ofcells could be loaded into a reaction volume. Concurrently, thetransport fluid contained therein is conducted into the waste channel38. The cell and its volume of suspension fluid in the reaction volumeis then transported along main microchannel 10 to the outlet of reagentchannel 28. The reagent 34 is pumped into the reaction volume containingthe cell, displacing the cell suspension fluid. The cell suspensionfluid is conducted away through waste channel 38′. The cell and reagentin the reaction volume are then transported to the detection/analysischannel 39. This process is repeated for each cell. Preferably, thecells 44′ are contained in reaction volumes between alternate sequentialpairs of segmenting fluid volumes as previously described herein.Cellular assays are examined as a function of time, pumping conditions,and media composition.

The extremely low consumption of reagent materials in microfluidic chipsprovides a significant advantage for screening expensive reagent andinhibitor materials and their substrate materials. Peptide libraries forsuch experiments can be custom synthesized on 10 to 100 micron diameteror larger beads with orthogonal releasable linkers. Shown in FIG. 6 is abasic arrangement for implementing a bead screening procedure on afluidic microchip in accordance with the present invention. A pair ofmicrochannels 10 and 610 are arranged in spaced parallel relation. Abuffer reservoir 80 is connected to the microchannel 610 through abuffer channel 82. A waste reservoir 84 is connected to the microchannel10 through a waste channel 86. A transfer channel 88 interconnects themicrochannel 610 to the microchannel 10 at a location substantially inalignment with the buffer channel 82 and the waste channel 86.

A series of reaction volumes are formed between segmenting fluid volumes26 a-26 d in microchannel 10 and a corresponding series of reactionvolumes are formed between segmenting fluid volumes 626 a-626 d inmicrochannel 610. The reaction volumes in each microchannel aretransported along the respective microchannels such that reactionvolumes b_(i−1), b_(i), and b_(i+1) in microchannel 610 are maintainedin synchronism with reaction volumes c_(j−1), c_(j), and c_(j+1) inmicrochannel 10. Beads 44 a, 44 b, and 44 c are inserted in the reactionvolumes b_(i−1), b_(i), and b_(i+1), respectively, in a manner similarto that described above with reference to FIG. 3. Enzyme volumes 36 a,36 b, and 36 c are inserted in the reaction volumes c_(j−1), c_(j), andc_(j+1), respectively, in a manner similar to that described above withreference to FIG. 2.

When each bead arrives at the transfer channel 88, its compound isreleased from the bead and transferred to the corresponding reactionvolume. The bead and its associated reaction volume are then shifted inregistration to a downstream station where a fluorogenic substrate isadded to the reaction volume (c_(j−1), c_(j), and c_(j+1)) forfluorescence assay. A bead corresponding to a reaction volume thatexhibits inhibition can be sorted out and transferred to a station wherea second release of compound is effected by an orthogonal cleavagemethod. That compound can be analyzed by electrospray ionization massspectrometry to determine its chemical structure.

The utilization of any of the fluidic microchip-implemented proceduresdescribed above yields an extended series of discrete assays, cells,beads, etc. transported in a microchannel. The reaction volumes involvedare preferably nanoliter or sub-nanoliter volumes, thus providingnumerous different compounds, assays, cells, beads, etc. It is alsocontemplated that larger volumes can be used. Since each reaction volumeis discrete, its position can be identified and tracked as it movesthrough the microchannel, analogous to a series of electronic bitsmoving through a digital shift register. Shown in FIG. 7 is anarrangement for archiving and retrieving numerous reaction volumescontaining reagents, cells, combinational library beads, etc. Amicrochannel 10 is connected to a source of transport fluid through atransport fluid channel 60. As described previously, a segmenting fluidis provided to the microchannel 10 through a branch channel 16.Reagents, cells, or beads are inserted into the reaction volumes inmicrochannel 10 through reagent channel 28. A waste channel 38 isinterconnected with microchannel 10 for conducting away the transportfluid from the reaction volume.

The microchannel 10 extends in one direction to form a plurality ofloops 64 a, 64 b, and 64 c and terminates in a reservoir 62 a.Microchannel 10 extends in an opposite direction to form a secondplurality of loops 66 a, 66 b, and 66 c, and terminates in a reservoir62 b. In a first mode of operation, the reaction volumes formed betweenthe segmenting fluid volumes are advanced by transporting them instep-wise fashion in the direction shown by the solid arrows and throughloops 64 a, 64 b, and 64 c for archiving. In a second mode of operation,the reaction volumes are retrieved by transporting them in step-wisefashion in the direction shown by the dashed arrows. The combined lengthof loops 66 a, 66 b, and 66 c is similar in length to the combinedlength of loops 64 a, 64 b, and 64 c, so that the entire series ofreaction volumes archived in loops 64 a, 64 b, and 64 c can be retrievedfor later analysis.

Although not shown in FIG. 7, the electrode layout needed to implementthe archiving/retrieval device would be such as to enable the transportof the reaction volumes in two directions. Preferably, the electrodes orcontacts would enter from each of the four sides of the loops. Theelectrodes geometry is selected to provide adequate contact with themicrochannel loops at a proper spatial periodicity. With such a layout,materials can be loaded serially into the register to be analyzedimmediately or at a later time. The materials can be delivered toreagent or assay stations as the reaction volumes are shifted back andforth between the microchannel loops.

The number of reaction volumes that can be held in the storage bankdefined by loops 64 a, 64 b, and 64 c can be estimated using Equation 1below.

$\begin{matrix}{N = {\frac{4L_{S}}{\left( {L_{R} + L_{I}} \right)}\left( {n - {\frac{n\left( {{2n} - 1} \right)}{2}\frac{L_{C}}{L_{S}}}} \right)}} & \left\{ {{Eq}.\mspace{14mu} 1} \right\}\end{matrix}$L_(S) is the length of one side of the outermost loop, L_(R) is thelength of the reaction volume, L_(I) is the length of the isolatingsegment, L_(C) is the center-to-center spacing between adjacent channelsin the loops, and n is the number of loops. For example, if L_(S) is 100mm, L_(R)+L_(I)=1 mm, L_(C) is 0.1 mm, and n=100, then approximately19010 reaction volumes (N) can be stored in the shift register storageloops. Such a device would use only about 10% of the area of a 100mm×100 mm square microchip substrate. Although the preferred arrangementdescribed herein includes one or more loops to form the storage channel,it will be readily appreciated by those skilled in the art that otherconfigurations can be utilized. For example, a serpentine arrangementwould be equally effective.

Such shift register storage/retrieval devices are very advantageous forhandling combinatorial bead libraries as described with reference toFIGS. 5 and 6. Beads from a split-synthesis could be collectively loadedinto a reservoir and dispensed, using on-chip bead sorting capabilities,into the storage register. Compounds can be partially photolyticallyreleased from the beads as needed or prereleased into the contents ofthe reaction volume. The bead library can be certified by incorporatingan electrospray ionization station on the microchip for massspectrometric identification of the reaction compounds. Beads can alsobe brought to a location where the compound is delivered to a separatereaction volume to perform a biological assay as described withreference to FIG. 6.

Referring now to FIG. 8, there is shown a system 800 for accomplishingthe identification of new drugs. A synthesis module 810 for synthesizinga series of potentially useful drug compounds is connected to a mainmicrochannel 812 through a plurality of insertion channels. Each of thesynthesized compounds is inserted into a transport volume andtransported along microchannel 812 in the manner described previouslyhereinabove. A certification module 814 is provided for analyzing andcertifying the molecular structure of the synthesized compounds. Thecertification module is connected to the microchannel 812 through asecond plurality of channels for obtaining samples of the synthesizedcompounds. An assay module 818 for performing screening assays on theseries of synthesized compounds against molecular or cellular targets isalso provided. Screening module 818 is connected to the microchannel 812by a third plurality of channels for obtaining samples of thesynthesized drug compounds. The target molecules or cells are stored inserial fashion in the target storage modules 820, 821. A secondmicrochannel 822 is disposed between the target storage modules fortransporting the targets to the screening module 818.

The series of drug compounds are transported along microchannel 812 tostorage modules 816, 817 for later retrieval. This feature permits thedrug compounds to be analyzed and/or screened at a time substantiallylater than the time of synthesis. The results of the screeningsperformed by the screening module 818 are provided to a decision module824 which can evaluate the effectiveness of the synthesized compoundsand provide feedback to the synthesis module 810 for synthesizing newand different compounds based on the earlier results. In this manner amultitude of new drug compounds can be rapidly and automaticallysynthesized, certified, and screened on a single microchip.

In view of the foregoing descriptions and the accompanying drawings, itwill be appreciated by those skilled in the art that the method andapparatus according to this invention are capable of addressing a broadrange of biochemical analysis problems that benefit from precise andautomated nanoliter or subnanoliter scale manipulations with high serialthroughput capacity. The device and method described herein also lendthemselves to multiple parallel expansion which will provide greaterthroughput for the generation of chemical and biochemical information.The microchannel devices described can manipulate biochemical reactionvolumes in a controlled manner to provide rapid results, e.g., rates ofat least about 1 to 10 Hz per channel, and rates up to 100 Hz or 1000 Hzare expected to be achievable. The reaction volumes utilized are capableof containing molecular or particulate species without diffusive losses.

The individual reaction volumes are manipulated in serial fashionanalogous to a digital shift register. The device includes loopedmicrochannels to provide serial storage of the reaction volumes forlater retrieval. The method and apparatus according to this inventionhave application to such problems as screening molecular or cellulartargets using single beads from split-synthesis combinatorial libraries,screening single cells for RNA or protein expression, genetic diagnosticscreening at the single cell level, or performing single cell signaltransduction studies.

The terms and expressions which have been employed in the foregoingdescription are used as terms of description and not of limitation.There is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized, however, that various modificationssuch as channel dimension, location, and arrangement are possible withinthe scope of the invention as claimed.

1. A method of forming and transporting a series of discrete nano- tosubnano-liter reaction volumes on a microfluidic microchip, comprising:providing a microfluidic microchip having formed therein a firstmicrochannel and a source of a first liquid, the first microchannelhaving a first end in liquid communication with the source of firstliquid and having a second end, and a second microchannel and a sourceof second liquid immiscible with the first liquid, the secondmicrochannel having a first end in liquid communication with the sourceof second liquid and a second end interconnected with the firstmicrochannel; providing a vacuum source in fluid communication with thesecond end of the first microchannel; and activating the vacuum sourceto simultaneously draw both a volume of the first liquid in the firstmicrochannel through the first end of the first microchannel and avolume of second liquid into the first microchannel through the secondend of the second microchannel to provide a series of discrete nano- tosubnano-liter volumes of the second liquid within the firstmicrochannel.
 2. The method of claim 1 wherein the first liquidcomprises paraffin oil, mineral oil, silicon oil, a perfluorocarbon, anonconducting fluid, a non-polar solvent, or combinations thereof. 3.The method of claim 1, wherein the second liquid is aqueous.